we did gene transformation to tomato plant by agrobacterium tumefaciens. but when we examined the obtained explants, PCR results were sometimes positive and sometimes negative when we examined the DNA sequence located between the RB and LB part of the binary vector. We believe that sometimes positive results are obtained because Agrobaterium remains in the plant body. How can it be determined that the T-DNA part has been integrated into the plant genome? No growth of negative control plants was observed in selective medium.
We performed the transformation using the following method.
Agrobacterium tumefaciens-Mediated Transformation of Tomato
Joyce Van Eck 1 2, Patricia Keen 3, Michelle Tjahjadi 3
Affiliations expand
- PMID: 30415340
- DOI: 10.1007/978-1-4939-8778-8_16
The PCR check can definitely be influenced by residual bacteria. If you have the reagents and equipment for a Southern blot that should work.
Amy Klocko. thank you very much for your advice.
but we don't have enough reagents for Southern blot. How can I clean my sample from Agrobacterium DNA?
can i try the method given in below article
The protocol in the article seems like a good place to start and try something, I haven't used that approach myself. Folks I work with use selection against the bacterium and PCR to detect the Agro itself (you want detection of your gene of interest and no detection of Agro genes, good controls are essential here).
Hi,
you could (should) check for persisting Agrobacteria. Mostly people do it by PCR using Agrobacteria-specific primers, like for the vir gene. The actual primer sequences might depend on the Agro-strain you used. The ones fitting your strain should be found in the literature.
Best of luck!
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